Streptococcus pneumoniae inhabits the human respiratory tract and causes disease when it leaves the nasopharyngeal region and invades normally sterile sites. Asymptomatic carriage is far more common than disease, yet these bacteria are important causes of otitis media, pneumonia, meningitis, and bacteremia in young children. Despite advances in our understanding of the epidemiology and pathogenesis of these bacteria, the precise factors that predispose a pneumococcal strain for carriage versus disease are not known. S. pneumoniae strains differ in their propensity to cause disease, and the genetic background of a particular strain is likely to influence its ability to successfully invade different tissue sites. We propose an interdisciplinary molecular and epidemiologic approach to increase understanding of the differences between pneumococcal carriage and clinical isolates. Our overall aims are 1) to determine the molecular epidemiology of S. pneumoniae clinical and carriage isolates from children <5 years of age;2) to identify tissue specific virulence factors associated with S. pneumoniae strains that cause clinical disease in children <5 years of age and evaluate their distribution among a large collection of clinical and carriage strains;and 3) to evaluate candidate otitis media specific virulence genes in the chinchilla model of otitis media. Specifically, we will determine the association of S. pneumoniae clones with carriage and specific clinical diseases using multilocus sequence typing, identify individual genetic determinants of tissue specific virulence using genomic subtraction, and evaluate the relative importance of these putative virulence determinants at the population level using a dot blot hybridization screen of a large, clinically relevant collection of pneumococcal isolates. These isolates are from healthy children and children with bacteremia, pneumonia, meningitis and otitis media. S. pnueumoniae deletion mutants in otitis media associated genes will be created and their virulence tested in an experimental chinchilla model of otitis media. A greater understanding of the genetic factors that influence the virulence potential of pneumococcal clones is critical to our ability to develop effective methods to control, prevent, and treat these important infections.